Presence and regulation of cannabinoid receptors in human retinal pigment epithelial cells.

PURPOSE
Cannabinoid receptors have been detected in neuron cells and proposed as potential therapeutic agents in neurodegenerative disorders because of their involvement in controlling neural cell survival and death. However, their presence and role in human retinal pigment epithelial (RPE) cells, which play a key role in initiating and developing age related macular degeneration (ARMD), have never been investigated. Here we analyzed the expression of and changes in cannabinoid receptors (CB1 and CB2) and one enzyme responsible for endocannabinoid hydrolysis, fatty acid amide hydrolase (FAAH), in RPE cell oxidative damage process, a cellular model of ARMD.


METHODS
Primary human RPE cells and cells from the ARPE-19 cell line were cultured and exposed to H2O2 for 24 h to induce oxidative damage. Real time RT-PCR, immunofluorescent staining, and western blot methods were performed to study the expression of and changes in CB1 and CB2 receptors, and FAAH. Cell viability and reactive oxygen species (ROS) production were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and a dichlorofluorescein (DCF) assay, respectively. PI3K/Akt and ERK1/2 protein expression and activation of signaling molecules were assessed by western blot analysis.


RESULTS
By using real time RT-PCR, immunofluorescent staining and western blot methods, we showed that human RPE cells express CB1, CB2, and FAAH. Meanwhile, oxidative stress can upregulate CB1 and CB2 receptor expression, and downregulate FAAH expression. The CB1/CB2 receptor agonist, CP55,940, and the CB2 receptor agonist, JWH015 significantly protected RPE cells from oxidative damage. In addition, CP55,940 significantly reduced the levels of intracellular ROS, strengthened oxidative stress-induced activation of PI3K/Akt and reduced activation of the ERK1/2 signal pathway.


CONCLUSIONS
The results demonstrate the expression and regulation of CB1 and CB2 receptors and FAAH in human RPE cells. The modulation of cannabinoid receptor tone warrants consideration for future therapeutic strategies of ARMD.

of cannabinoid receptors in retinal neurodegenerative disorders have been studied far less than those in brain and other organ systems.
Age-related macular degeneration (ARMD) is a lateonset neurodegenerative retinal disease, which predominates in the elderly as a cause of irreversible and profound vision loss [9]. ARMD shares many common clinical and pathogenetic properties with other neurodegenerative disorders. Currently, there is no ideal therapy to prevent or cure ARMD. Intake of antioxidants is the only established treatment for those with the atrophic or "dry" form of ARMD [10]. Although treatment options for those with the neovascular or "wet" form have increased recently, even these treatments are largely ineffective for reversing existing visual impairment [11]. Thus, the development of strategies for preventing ARMD assumes great importance; and new treatment approaches should focus on the initial insults that lead to the disease's progression. ARMD initial pathogenesis includes degeneration, dysfunction, or loss of RPE cells caused by oxidative injury [12]. chromatography and mass spectrometry [13][14][15]. CB1 receptor, a cannabinoid receptor enriched in neuronal tissue, was localized in the inner and outer plexiform layers of a human retina [16]. Unlike CB1 receptor, CB2 receptor and FAAH were undetectable in the human retina, but were found in rat retina, mostly in the ganglion cells and in the soma of dopamine amacrine cells and large cells [17,18]. The function of ECS in the eye, in addition to regulating photoreception and neurotransmission in the retina, and the effects on intraocular pressure and ocular blood vessels [19][20][21][22][23][24], also encompasses neuroprotective effects against retinal neurotoxicity [25,26]. This background suggests a functional ECS in the retina. Eyes of patients with ARMD have been show increase levels of endocannabinoids (AEA) in their retina [27]. Therefore, we hypothesized that ECS, acting as a novel signaling system in the retina, may play a substantive role in the ARMD pathophysiological process. Because most effects of endocannabinoids are mediated by binding to cannabinoid receptors and terminated by degradation enzyme FAAH [28]. Thus, the present study aims to examine the expression of CB1 and CB2 receptors, and FAAH in human RPE cells, and their changes in oxidative stress conditions, using a cellular model of ARMD [29]. Our findings may set the basis for the potential pharmacological modulation of cannabinoid receptor tone as a novel therapeutic target in ARMD.

Primary human RPE cells and ARPE-19 cell line culture:
Human RPE cells were obtained from eye bank donor eyes, which were cut across the posterior pole, and the vitreous and neural retina were removed. Eyes were obtained from three male human donors between 30 and 50 years of age. None of the donors had a known history of eye disease. The eyes at time of receipt were all integral. RPE cells were isolated within 4 to 16 h after death.The remaining eyecup was washed with phosphate buffered saline (PBS, 8.00 g NaCl, 0.20 g KCl, 0.24 g KH2PO4 and 1.44 g Na2HPO4 in 1 l distilled water, pH 7.4) and incubated with 0.025% trypsin-ethylene diamine tetraacetic acid (EDTA; Invitrogen-Gibco, Carlsbad, CA) in a humidified chamber at 37 °C. The cells then were scraped gently and seeded in Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco) containing 15% fetal bovine serum (FBS; Invitrogen-Gibco).
A human retinal pigment epithelial cell line (ARPE- 19) was purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were maintained in DMEM/F12 medium (Invitrogen-Gibco) supplemented with 10% FBS (Invitrogen-Gibco) and 1% penicillin and streptomycin, and were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed every two days. The ARPE-19 cells were used within 10 generations. Since cells of lower generations were quite sensitive to oxidative stress. For each concentration of H2O2 and the compounds, five wells were analyzed. Each experiment was performed at least three times.
RNA extraction: Total RNA was isolated from primary human RPE cells, ARPE-19 cells and, H2O2 (200 μM)-treated ARPE-19 cells using the RNeasy Total RNA System (RNeasy Mini Kit, Qiagen, Valencia, CA) following the manufacturer's recommendation, and then treated with RNase-free DNase I to remove any contaminating genomic DNA. The isolated RNA had optic density (OD) 260/280 ratios greater than or equal to 2.0 [30]. To synthesize a cDNA template for PCR, we reverse transcribed 1 μg of total RNA with oligo-(dT) primer and reverse transcriptase (ReverTra Ace, Toyobo Co., Ltd., Osaka, Japan). The quality of first-strand cDNA was confirmed by PCR with β-actin primers.

Real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR):
Real-time RT-PCR was performed for quantitative analysis according to the standard protocol using the SYBR Green PCR Master Mix (Toyobo Co., Ltd., Osaka, Japan). PCR conditions for CB1 and CB2 were as follows: after initial denaturation at 95 °C for 5 min, 40 cycles were performed at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, followed by a 10 min extension at 72 °C. For FAAH, the conditions were as follows: after initial denaturation at 95 °C for 5 min, 40 cycles were performed at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 1 min, followed by a 10 min extension at 72 °C.
Primers used were shown in Table 1. Quantification analysis of CB1, CB2, and FAAH mRNA was normalized with β-actin as reference. The specificity of PCR amplification products was checked by performing a dissociation melting curve analysis. Relative multiples of changes in mRNA expression were determined with the relative comparative threshold (CT) method.
Immunofluorescent staining: CB1 and CB2 expression in the ARPE-19 cells was determined by immunofluorescent staining. In brief, ARPE-19 cells were grown to confluence in chamber slides (Nalgene-Nunc, Lab-Tek, Naperville, IL). The growth medium was aspirated and the cells were washed three times with PBS, and then fixed with 4.0% paraformaldehye for 20 min at 4 °C. After the cells had been washed with PBS, they were permeabilized with 0.2% Triton X-100 in PBS for 15 min at room temperature. Subsequently, CB1 and CB2 expression in the ARPE-19 cells was determined by immunofluorescent staining using a 1:100 dilution of anti-CB1 (rabbit polyclonal, Abcam, Cambridge, UK) or anti-CB2 (rabbit polyclonal, Abcam), respectively, for 6 h at 4 °C. After the cells had been rinsed with PBS, they were probed with 1:250 goat anti-rabbit FITC (Pierce, Rockford, IL) for 1 h at room temperature. Their nucleus was counterstained with 4', 6-diamidino-2-phenylindole (Molecular Probes, Invitrogen-Gibco, Carlsbad, CA). Images were obtained with a fluorescent microscope (Olympus IX 81) at 20X objective with 1.5X optical zoom (Olympus IX 81, Olympus Optical, Tokyo, Japan). MTT assay for cell viability: An 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) assay is a qualitative index of cell viability. Mitochondrial and cytosolic dehydrogenases of living cells reduce the yellow tetrazolium salt (MTT) to produce a purple formazan dye that can be detected spectrophotometrically [31]. After ARPE-19 cells were preincubated with various concentrations of CP55,940, ACEA, and JWH015 for 15 min followed by exposure to 200 μM H2O2 for 24 h. Then, MTT (Sigma, St. Louis, MO) was added to a final concentration of 0.5 mg/ml and incubated for 4 h at 37 °C. The culture medium was then removed and the remaining blue precipitate was solubilized in dimethyl sulfoxide, followed by reading absorbance at 570 nm in a plate reader using 630 nm as a reference (Spectra Max 340; Molecular Devices, Sunnyvale, CA). This reading was divided by the adjusted absorbance reading of untreated cells in control wells to obtain the percentage of cellular survival. Reactive oxygen species determination: The intracellular reactive oxygen species (ROS) level is an important biomarker for oxidative stress. An increased ROS level generally indicates increased oxidative stress. Relative ROS production was determined by the formation of a fluorescent Dichlorofluorescein (DCF) compound on oxidation of the nonfluorescent, reduced DCF-DA [32]. Cells were incubated with 10 μM DCF-DA at 37 °C for 30 min, and then washed twice with PBS. Relative fluorescence was measured using a fluorescence plate reader at 485 nm excitation and 535 nm emission wavelength (Wallace, Perkin-Elmer, Warrington, UK).

Effect of H2O2 on ARPE-19 cell viability:
The MTT assay for cell viability was used to quantify the ARPE-19 cell cytotoxic response to H2O2. H2O2 produced a progressive, cytotoxic effect in the ARPE-19 cells, beginning at a dose of 100 μM, with 5.7% cytotoxicity and reaching a maximum toxicity at 77.5% damaged cells measured at 500 μM after 24 h of incubation (p<0.05; Figure 1). The results indicate that treating ARPE-19 cells with H2O2 for 24 h caused a dosedependent decrease in their viability with the half maximal inhibitory concentration (IC50) of 200 μM. Figure 2A shows, real time RT-PCR revealed primary human RPE cells expressed CB1, CB2, and FAAH mRNA. ARPE-19 cells were treated with 200 μM H2O2 for 24 h, and the changes in CB1, CB2, and FAAH mRNA expression were determined by quantitative RT-PCR.

DISCUSSION
To the best of our knowledge, this is the first demonstration of the presence of CB1, CB2, and FAAH in human RPE cells, which play a key role in ARMD initiation and development. Plenty of evidence in the previous literature suggests that oxidative stress is a contributing factor to RPE dysfunction in ARMD [33]. The results led us to further analyze the cannabinoid receptors and FAAH expression changes in RPE cells induced by oxidative stress. Most studies have used human RPE cells as the in vitro study model of ARMD, either primary or cell line. One of the drawbacks of using primary RPE cells is that it provides a limited number of cells and may lack a consistent cellular background. The ARPE-19 cell line overcomes this problem while maintaining the morphological and functional characteristics of primary cells. In addition, ARPE-19 cells respond to oxidative stress (H2O2, media starvation, etc.) in a fashion similar to that of primary cultured human RPE cells. Therefore, we have used the ARPE-19 cell line for most of our studies. H2O2 added to the culture medium was used as a chemical oxidant. This agent is convenient and biologically relevant, especially for the RPE cells. H2O2 has been found in ocular tissues in vivo [34] and can be produced by the RPE cells as a reactive oxygen intermediate during photoreceptor outer segment phagocytosis [35]. For these reasons, we selected H2O2 for our studies and performed a series of dose response assays to determine the working concentration that led to a consistent and reliable cytotoxicity.
By using real-time RT-PCR and the western blot method, we showed that oxidative stress can upregulate CB1 and CB2 receptor expression and downregulate FAAH expression in a cell model of ARMD. These results agree with a previous comparative analytical study, which showed that endocannabinoids (AEA) increased in the retina from human ARMD eyes [27]. As a lipid soluble substance, AEA cannot be stored in vesicles; and therefore it is synthesized on demand and travels, in a retrograde direction, across the postsynaptic membrane to the presynaptic membrane, where it activates presynaptic CB1 and CB2 receptors [36]. After cellular uptake, AEA is degraded via the enzyme FAAH [14]. Variation of ECS also can be observed in other neurodegenerative disorders, such as Parkinson disease, Alzheimer disease, and multiple sclerosis [2,37,38]. The variation of ECS may be an endogenous response to maintain endocannabinoid homeostasis and regulate the pathologic function of neuron cells [39,40]. In line with the endocannabinoid homeostasis theory, there are now several examples of the successful use of ECS-directed drugs used to alleviate the clinical symptoms of neurodegenerative diseases in animal models [2,41,42]. These results suggest that cannabinoid receptors may be potential targets for therapeutic interventions for ARMD.
We also introduced CP55,940, which is a nonselective CB1/CB2 receptor agonist in oxidative stress-induced RPE cellular damage. We found that CP55,940 protected ARPE-19 cells from oxidative stress-induced cell damage and intracellular ROS generation in a dose-dependent way with excellent efficacy. We further explored whether CP55,940 induced cytoprotective signaling pathways while rescuing RPE cells from oxidative damage. The PI3K/Akt-and ERK1/2-mediated survival signal pathways had been suggested to protect RPE cells from oxidative stress [43,44]. We therefore addressed whether CP55,940 could modulate PI3K/Akt and ERK1/2 pathways, and found that CP55,940 could enhance H2O2-induced activation of PI3K/Akt and reduce the activation of the ERK1/2 pathway. We further investigated whether the selective activation of CB1 or CB2 receptor could protect RPE cells from oxidative stress induced damage. We used a cell viability assay to determine viability in cells treated with H2O2. We demonstrated that CB2 receptor agonist, but not CB1 receptor agonist, significantly protected human RPE cells from oxidative stress. Several studies have shown CB1 receptor activation to mediate neuroprotection, but this is not a universal finding [45,46]. This neuroprotective effect could be related to different cell types and pathological conditions.
In conclusion, we demonstrated the expression of CB1 and CB2 receptors and FAAH in human RPE cells, and their changes in oxidative stress conditions. The RPE cells perform vital functions for safeguarding photoreceptor cells against oxidative stress and are involved in the pathogenesis of ARMD [47]. These findings open up the attractive possibility that a correlation exists between endocannabinoid homeostasis and the onset of ARMD. However, as we observe inhibition, rather than the more usual stimulation, of ERK signaling, the involvement of receptors other than CB1 as well as CB2 in the effects of CP55,940 or JWH015 cannot be excluded. More studies using siRNA or specific CB1 and CB2 receptor antagonists should be performed to examine whether manipulating the levels of cannabinoid receptors could be a novel pharmacological approach to treat ARMD in the future.